ImprintSeq, a novel tool to interrogate DNA methylation at human imprinted regions and diagnose multilocus imprinting disturbance.

PURPOSE: Disruptions of genomic imprinting are associated with congenital imprinting disorders (CIDs) and other disease states, including cancer. CIDs are most often associated with altered methylation at imprinted differentially methylated regions (iDMRs). In some cases, multiple iDMRs are affected causing multilocus imprinting disturbances (MLIDs). The availability of accurate, quantitative, and scalable high-throughput methods to interrogate multiple iDMRs simultaneously would enhance clinical diagnostics and research. METHODS: We report the development of a custom targeted methylation sequencing panel that covered most relevant 63 iDMRs for CIDs and the detection of MLIDs. We tested it in 70 healthy controls and 147 individuals with CIDs. We distinguished loss and gain of methylation per differentially methylated region and classified high and moderate methylation alterations. RESULTS: Across a range of CIDs with a variety of molecular mechanisms, ImprintSeq performed at 98.4% sensitivity, 99.9% specificity, and 99.9% accuracy (when compared with previous diagnostic testing). ImprintSeq was highly sensitive for detecting MLIDs and enabled diagnostic criteria for MLID to be proposed. In a child with extreme MLID profile a probable genetic cause was identified. CONCLUSION: ImprintSeq provides a novel assay for clinical diagnostic and research studies of CIDs, MLIDs, and the role of disordered imprinting in human disease states.

Novel variant in the CNNM2 gene associated with dominant hypomagnesemia.

The maintenance of magnesium (Mg2+) homeostasis is essential for human life. The Cystathionine-ß-synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) have been described to be involved in maintaining Mg2+ homeostasis. Among these CNNMs, CNNM2 is expressed in the basolateral membrane of the kidney tubules where it is involved in Mg2+ reabsorption. A total of four patients, two of them with a suspected disorder of calcium metabolism, and two patients with a clinical diagnosis of primary tubulopathy were screened for mutations by Next-Generation Sequencing (NGS). We found one novel likely pathogenic variant in the heterozygous state (c.2384C>A; p.(Ser795*)) in the CNNM2 gene in a family with a suspected disorder of calcium metabolism. In this family, hypomagnesemia was indirectly discovered. Moreover, we observed three novel variants of uncertain significance in heterozygous state in the other three patients (c.557G>C; p.(Ser186Thr), c.778A>T; p.(Ile260Phe), and c.1003G>A; p.(Asp335Asn)). Our study shows the utility of Next-Generation Sequencing in unravelling the genetic origin of rare diseases. In clinical practice, serum Mg2+ should be determined in calcium and PTH-related disorders.

Forty-One Individuals With Mutations in the AVP-NPII Gene Associated With Familial Neurohypophyseal Diabetes Insipidus.

CONTEXT: Familial neurohypophyseal diabetes insipidus is a rare disease produced by a deficiency in the secretion of antidiuretic hormone and is caused by mutations in the arginine vasopressin gene. OBJECTIVE: Clinical, biochemical, and genetic characterization of a group of patients clinically diagnosed with familial neurohypophyseal diabetes insipidus, 1 of the largest cohorts of patients with protein neurophysin II (AVP-NPII) gene alterations studied so far. DESIGN: The AVP-NPII gene was screened for mutations by PCR followed by direct Sanger sequencing in 15 different unrelated families from Spain. RESULTS: The 15 probands presented with polyuria and polydipsia as the most important symptoms at the time of diagnosis. In these patients, the disease was diagnosed at a median of 6 years of age. We observed 11 likely pathogenic variants. Importantly, 4 of the AVP-NPII variants were novel (p.(Tyr21Cys), p.(Gly45Ser), p.(Cys75Tyr), p.(Gly88Cys)). CONCLUSIONS: Cytotoxicity seems to be due to consequences common to all the variants found in our cohort, which are not able to fold correctly and pass the quality control of the ER. In concordance, we found autosomal dominant familial neurohypophyseal diabetes insipidus in the 15 families studied.

Novel mutations associated with inherited human calcium-sensing receptor disorders: A clinical genetic study.

Objective Molecular diagnosis is a useful diagnostic tool in calcium metabolism disorders. The calcium-sensing receptor (CaSR) is known to play a central role in the regulation of extracellular calcium homeostasis. We performed clinical, biochemical and genetic characterization of sequence anomalies in this receptor in a cohort of 130 individuals from 82 families with suspected alterations in the CASR gene, one of the largest series described. Methods The CASR gene was screened for mutations by polymerase chain reaction followed by direct Sanger sequencing. Results Presumed CaSR-inactivating mutations were found in 65 patients from 26 families. These patients had hypercalcemia (median: 11.3 mg/dL) but normal or abnormally high parathyroid hormone (PTH) levels (median: 52 pg/mL). On the other hand, presumed CaSR-activating mutations were detected in 17 patients from eight families. These patients had a median serum calcium level of 7.4 mg/dL and hypoparathyroidism (median: PTH 13 pg/mL). Further, common polymorphisms previously associated with high blood ionized calcium levels were found in 27 patients (median calcium: 10.6 mg/dL; median PTH: 65 pg/mL) with no other alterations in CASR. Overall, we found 30 different mutations, of which, 14 have not been previously reported (p.Ala26Ser, p.Cys60Arg, p.Lys119Ile, p.Leu123Met, p.Glu133Val, p.Gly222Glu, p.Phe351Ile, p.Cys542Tyr, p.Cys546Gly, p.Cys677Tyr, p.Ile816Val, p.Ala887Asp, p.Glu934*, p.Pro935_Gln945dup). Conclusions Patients with CASR mutations may not fit the classic clinical pictures of hypercalcemia with hypocalciuria or hypocalcemia with hypercalciuria. Molecular studies are important for confirming the diagnosis and distinguishing it from other entities. Our genetic analysis confirmed CaSR disorders in 82 patients in the study cohort.

Identification of a novel large CASR deletion in a patient with familial hypocalciuric hypercalcemia.

Familial hypocalciuric hypercalcemia type I is an autosomal dominant disorder caused by heterozygous loss-of-function mutations in the CASR gene and is characterized by moderately elevated serum calcium concentrations, low urinary calcium excretion and inappropriately normal or mildly elevated parathyroid hormone (PTH) concentrations. We performed a clinical and genetic characterization of one patient suspected of familial hypocalciuric hypercalcemia type I. Patient presented persistent hypercalcemia with normal PTH and 25-hydroxyvitamin D levels. The CASR was screened for mutations by PCR followed by direct Sanger sequencing and, in order to detect large deletions or duplications, multiplex ligation-dependent probe amplification (MLPA) was used. One large deletion of 973 nucleotides in heterozygous state (c.1733-255_2450del) was detected. This is the first large deletion detected by the MLPA technique in the CASR gene. Learning points: Molecular studies are important to confirm the differential diagnosis of FHH from primary hyperparathyroidism. Large deletions or duplications in the CASR gene can be detected by the MLPA technique. Understanding the functional impact of the mutations is critical for leading pharmacological research and could facilitate the therapy of patients.

Poor phenotype-genotype association in a large series of patients with Type III Bartter syndrome.

INTRODUCTION: Type III Bartter syndrome (BS) is an autosomal recessive renal tubule disorder caused by loss-of-function mutations in the CLCNKB gene, which encodes the chloride channel protein ClC-Kb. In this study, we carried out a complete clinical and genetic characterization in a cohort of 30 patients, one of the largest series described. By comparing with other published populations, and considering that 80% of our patients presented the p.Ala204Thr Spanish founder mutation presumably associated with a common phenotype, we aimed to test the hypothesis that allelic differences could explain the wide phenotypic variability observed in patients with type III BS. METHODS: Clinical data were retrieved from the referral centers. The exon regions and flanking intronic sequences of the CLCNKB gene were screened for mutations by polymerase chain reaction (PCR) followed by direct Sanger sequencing. Presence of gross deletions or duplications in the region was checked for by MLPA and QMPSF analyses. RESULTS: Polyuria, polydipsia and dehydration were the main common symptoms. Metabolic alkalosis and hypokalemia of renal origin were detected in all patients at diagnosis. Calciuria levels were variable: hypercalciuria was detected in 31% of patients, while 23% had hypocalciuria. Nephrocalcinosis was diagnosed in 20% of the cohort. Two novel CLCNKB mutations were identified: a small homozygous deletion (c.753delG) in one patient and a small deletion (c.1026delC) in another. The latter was present in compound heterozygosis with the already previously described p.Glu442Gly mutation. No phenotypic association was obtained regarding the genotype. CONCLUSION: A poor correlation was found between a specific type of mutation in the CLCNKB gene and type III BS phenotype. Importantly, two CLCNKB mutations not previously described were found in our cohort.

A Novel Mutation in a Patient with Hyperparathyroidism-Jaw Tumour Syndrome.

Hyperparathyroidism-jaw tumour syndrome (HPT-JT) is a rare variant of familial hyperparathyroidism, characterized by primary hyperparathyroidism (PHPT) due to one or multiple parathyroid adenomas, and benign tumours of the mandible and maxilla. It has an autosomal dominant pattern of inheritance, and is associated with mutations that deactivate the cell division cycle protein 73 homolog (CDC73) gene, also known as hyperparathyroidism 2 (HRPT2), located on the long arm of chromosome 1, that encodes for the tumour suppressor protein parafibromin. In the majority of cases, PHPT is the presenting symptom, but up to 30 % of HPT-JT cases initially present with an ossifying fibroma of the maxillofacial bones. HPT-JT may result in severe hypercalcemia-related complications and an elevated risk of parathyroid carcinoma. For this reason, early identification of the disease is important. We present the case of a 23-year-old woman who was found to have jaw tumours and was later diagnosed with PHPT. Genetic analysis revealed a novel mutation in exon 1 of CDC73. This report contributes to the understanding of the genetics of this rare syndrome. It also highlights the fact that HPT-JT should be considered and CDC73 mutation analysis should be performed in cases of early-onset PHPT associated with ossifying fibromas of the jaw.

Novel mutations associated with nephrogenic diabetes insipidus. A clinical-genetic study.

UNLABELLED: Molecular diagnosis is a useful diagnostic tool in primary nephrogenic diabetes insipidus (NDI), an inherited disease characterized by renal inability to concentrate urine. The AVPR2 and AQP2 genes were screened for mutations in a cohort of 25 patients with clinical diagnosis of NDI. Patients presented with dehydration, polyuria-polydipsia, failure to thrive (mean ± SD; Z-height -1.9 ± 2.1 and Z-weight -2.4 ± 1.7), severe hypernatremia (mean ± SD; Na 150 ± 10 mEq/L), increased plasma osmolality (mean ± SD; 311 ± 18 mOsm/Kg), but normal glomerular filtration rate. Genetic diagnosis revealed that 24 male patients were hemizygous for 17 different putative disease-causing mutations in the AVPR2 gene (each one in a different family). Of those, nine had not been previously reported, and eight were recurrent. Moreover, we found those same AVPR2 changes in 12 relatives who were heterozygous carriers. Further, in one female patient, AVPR2 gene study turned out to be negative and she was found to be homozygous for the novel AQP2 p.Ala86Val alteration. CONCLUSION: Genetic analysis presumably confirmed the diagnosis of nephrogenic diabetes insipidus in every patient of the studied cohort. We emphasize that we detected a high presence (50 %) of heterozygous females with clinical NDI symptoms. WHAT IS KNOWN: • In most cases (90 %), inherited nephrogenic diabetes insipidus (NDI) is an X-linked disease, caused by mutations in the AVPR2 gene. • In rare occasions (10 %), it is caused by mutations in the AQP2 gene. What is new: • In this study, we report 10 novel mutations associated with NDI. • We have detected a high presence (50 %) of heterozygous carriers with clinical NDI symptoms.

Novel ATP6V0A4 mutation described in a Tunisian patient with distal renal tubular acidosis.

UNLABELLED: Few data regarding molecular diagnosis of primary distal renal tubular acidosis (DRTA) in Tunisian population are available. CASE REPORT: 25-day-old male patient from consanguineous parents of Tunisian origin diagnosed with DRTA and without hearing impairment observed later in life. ATP6V0A4 gene sequencing demonstrated a novel homozygous G deletion in exon 13 (c.1221delG, p.Met408CysfsX10), leading to a premature stop codon. CONCLUSION: A novel ATP6V0A4 gene mutation confirmed autosomal recessive DRTA with normal hearing in the patient. Molecular analysis may help to rapidly diagnose autosomal recessive DRTA in Tunisian population.

Molecular diagnosis of distal renal tubular acidosis in Tunisian patients: proposed algorithm for Northern Africa populations for the ATP6V1B1, ATP6V0A4 and SCL4A1 genes.

BACKGROUND: Primary distal renal tubular acidosis (dRTA) caused by mutations in the genes that codify for the H + -ATPase pump subunits is a heterogeneous disease with a poor phenotype-genotype correlation. Up to now, large cohorts of dRTA Tunisian patients have not been analyzed, and molecular defects may differ from those described in other ethnicities. We aim to identify molecular defects present in the ATP6V1B1, ATP6V0A4 and SLC4A1 genes in a Tunisian cohort, according to the following algorithm: first, ATP6V1B1 gene analysis in dRTA patients with sensorineural hearing loss (SNHL) or unknown hearing status. Afterwards, ATP6V0A4 gene study in dRTA patients with normal hearing, and in those without any structural mutation in the ATP6V1B1 gene despite presenting SNHL. Finally, analysis of the SLC4A1 gene in those patients with a negative result for the previous studies. METHODS: 25 children (19 boys) with dRTA from 20 families of Tunisian origin were studied. DNAs were extracted by the standard phenol/chloroform method. Molecular analysis was performed by PCR amplification and direct sequencing. RESULTS: In the index cases, ATP6V1B1 gene screening resulted in a mutation detection rate of 81.25%, which increased up to 95% after ATP6V0A4 gene analysis. Three ATP6V1B1 mutations were observed: one frameshift mutation (c.1155dupC; p.Ile386fs), in exon 12; a G to C single nucleotide substitution, on the acceptor splicing site (c.175-1G > C; p.?) in intron 2, and one novel missense mutation (c.1102G > A; p.Glu368Lys), in exon 11. We also report four mutations in the ATP6V0A4 gene: one single nucleotide deletion in exon 13 (c.1221delG; p.Met408Cysfs*10); the nonsense c.16C > T; p.Arg6*, in exon 3; and the missense changes c.1739 T > C; p.Met580Thr, in exon 17 and c.2035G > T; p.Asp679Tyr, in exon 19. CONCLUSION: Molecular diagnosis of ATP6V1B1 and ATP6V0A4 genes was performed in a large Tunisian cohort with dRTA. We identified three different ATP6V1B1 and four different ATP6V0A4 mutations in 25 Tunisian children. One of them, c.1102G > A; p.Glu368Lys in the ATP6V1B1 gene, had not previously been described. Among deaf since childhood patients, 75% had the ATP6V1B1 gene c.1155dupC mutation in homozygosis. Based on the results, we propose a new diagnostic strategy to facilitate the genetic testing in North Africans with dRTA and SNHL.

Genetics of type III Bartter syndrome in Spain, proposed diagnostic algorithm.

The p.Ala204Thr mutation (exon 7) of the CLCNKB gene is a "founder" mutation that causes most of type III Bartter syndrome cases in Spain. We performed genetic analysis of the CLCNKB gene, which encodes for the chloride channel protein ClC-Kb, in a cohort of 26 affected patients from 23 families. The diagnostic algorithm was: first, detection of the p.Ala204Thr mutation; second, detecting large deletions or duplications by Multiplex Ligation-dependent Probe Amplification and Quantitative Multiplex PCR of Short Fluorescent Fragments; and third, sequencing of the coding and flanking regions of the whole CLCNKB gene. In our genetic diagnosis, 20 families presented with the p.Ala204Thr mutation. Of those, 15 patients (15 families) were homozygous (57.7% of overall patients). Another 8 patients (5 families) were compound heterozygous for the founder mutation together with a second one. Thus, 3 patients (2 siblings) presented with the c. -19-?_2053+? del deletion (comprising the entire gene); one patient carried the p.Val170Met mutation (exon 6); and 4 patients (3 siblings) presented with the novel p.Glu442Gly mutation (exon 14). On the other hand, another two patients carried two novel mutations in compound heterozygosis: one presented the p.Ile398_Thr401del mutation (exon 12) associated with the c. -19-?_2053+? del deletion, and the other one carried the c.1756+1G>A splice-site mutation (exon 16) as well as the already described p.Ala210Val change (exon 7). One case turned out to be negative in our genetic screening. In addition, 51 relatives were found to be heterozygous carriers of the described CLCNKB mutations. In conclusion, different mutations cause type III Bartter syndrome in Spain. The high prevalence of the p.Ala204Thr in Spanish families thus justifies an initial screen for this mutation. However, should it not be detected further investigation of the CLCNKB gene is warranted in clinically diagnosed families.

A founder mutation in the CLCNKB gene causes Bartter syndrome type III in Spain.

The term "Bartter syndrome" encompasses a group of closely related inherited tubulopathies characterized by markedly reduced NaCl transport by the distal nephron. At present, five different genetic variants have been demonstrated. The majority of patients with so-called classic Bartter syndrome carry inactivating mutations of the CLCNKB gene encoding the basolateral ClC-Kb chloride channel (Bartter syndrome type III). The purpose of this study was to investigate the underlying mutation in cases of classic Bartter syndrome followed at our center. Ten patients, including two sisters, with clinical and biochemical features of classic Bartter syndrome were included in the mutational analysis. They originated from different regions of Spain with either Basque or Spanish ancestry. There was no history of consanguineous marriage in any of the kindreds. The parents and siblings of each patient, as well as a population of 300 healthy control adult subjects, were also analyzed. All ten patients were found to be homozygous for an identical missense mutation in the CLCNKB gene, substituting a threonine for an alanine at codon 204 (A204T) in the putative fifth transmembrane domain of the protein. None of the 300 control subjects were homozygous for the A204T allele. Overall, the A204T mutation was detected on 2/600 control chromosomes. Despite sharing a common mutation, the clinical manifestations of the syndrome in the patients varied from lack of symptoms to severe growth retardation. Demonstration of a point mutation within the CLCNKB gene as the apparently unique cause of Bartter syndrome type III in Spain is highly suggestive of a founder effect. Our results also support the lack of correlation between genotype and phenotype in this disease.

Analysis of the expression of MICA in small intestinal mucosa of patients with celiac disease.

The MHC class I chain-related A gene (MICA) is expressed in gastrointestinal epithelium and functions as an immune activation signal under stress conditions. MICA protein binds to NKG2D, a receptor of gamma delta T cells containing the TCR variable region V(delta)1, which are the most abundant subset of T cells in the intestinal epithelium. Ingested gluten in patients with celiac disease (CD) may function as a stress signal for the epithelial cells, and could enhance MICA expression on their surface. In this study, we have analyzed MICA expression in intestinal biopsy specimens from newly diagnosed and treated CD patients and controls. Quantitative RT-PCR analysis did not show differences in MICA expression among the three groups. With these results, we conclude that overexpression of MICA does not seem to play an important role in the pathogenesis of CD, at least at the time of diagnosis.